alpha1-Adrenergic receptors (ARs) play important roles in control of blood pressure, nasal congestion, muscle growth, and other processes. Two native subtypes (alpha1A and alpha1B) can be distinguished pharmacologically, and three subtypes have been cloned (alpha1B, alpha1C, and alpha1D). Although one of the cloned subtypes (alpha1D) was originally thought to encode the pharmacologically defined alpha1A subtype, recent data suggests that this is unlikely. The relationship between the cloned receptors and the native subtypes must be understood, and any additional cDNA clones obtained, before the biochemical and functional roles of alpha1-AR subtypes can be clearly understood. Human SKNMC neuroepithelioma cells express at least two alpha1-ARs, resembling the alpha1A, and alpha1B subtypes. We have isolated, expressed, and partially characterized a full length chimeric genomic/cDNA clone from this cell line which is homologous to the rat alpha1D. We will use sense expression and antisense knockout of this and other human clones in SKNMC cells to clarify the relationship of the known clones to the pharmacological alpha1A subtype. If they are unrelated, as expected, we will obtain a full length alpha1A cDNA by expression cloning. With these clones we will be able to clearly define the drug specificities, signalling mechanisms, and interactions of alpha1-AR subtypes. Our major goals include: 1. To clearly define the relationship between the 3 known clones and the pharmacologically defined alpha1A subtype; 2. To obtain a full-length cDNA for the alpha1A subtype by expression cloning; 3. To determine whether different alpha1-AR receptor subtypes preferentially couple to different second messenger systems; and 4. To determine whether different subtypes co-existing in the same cell have additive, synergistic or redundant interactions which may contribute to the confusion about the number and properties of alpha1-AR subtypes. These experiments will clarify the number, signalling mechanisms, and interactions of alpha1-AR subtypes. Since drugs acting on these receptors have considerable therapeutic importance, such information may have direct clinical impact.